This proposal is concerned with the nature and the physiological significance of high density lipoprotein (HDL) binding sites, and has three major objectives: 1) to study the mechanism by which treatment of HDL with tetranitromethane (TNM) inhibits its binding to HDL specific binding sites. 2) to study the dependence of specific HDL binding on the lipid composition of membranes and 3) to elucidate the role of HDL binding in the transfer of free cholesterol (FC) and cholesteryl esters (CE) between HDL and cells. To study the mechanism of inhibition by TNM of HDL binding, we will prepare a reconstituted HDL in which the native phospholipids have been replaced by dimyristoylphosphatidylcholine (DMPC). The effect of nitration on the binding of the reconstituted HDL will be studied. To determine the importance of apoprotein crosslinking the effect of diethyl suberimidate treatment of HDL on binding will also be studied. In order to investigate the role of membrane lipids in the binding of HDL, plasma membranes from control cells and cells with increased HDL binding (human skin fibroblasts, rat ovarian cells and Fu5AH rat hepatoma cells) will be isolated. The relationship between HDL binding and lipid composition will be investigated. In addition, the lipid composition of isolated membranes will be changed by in vitro manipulation and the effect of this change on HDL binding will be studied. To study the role of HDL binding in the transfer of FC and CE between HDL and cells, the effect of inhibition of specific binding on these processes will be studied. In studies on the relationship between binding and FC transfer, human fibroblasts, Fu5AH rat hepatoma cells, and established methods for measuring the bidirectional flux of FC will be employed. HDL binding will be inhibited by nitration of HDL or bby FAB fragments raised against HDL. In studies on the relationship between binding and CE transfer an HDL reconstituted with cholesteryl ether, and primary cultures of rat hepatocytes and ovarian granulosa cells will be employed. The binding of reconstituted HDL will be inhibited by the Fab fragments. The results of the proposed studies will contribute to a better understanding of the antiatherogenic properties of HDL.